For IHC staining, the sections were dewaxed 3 times with xylene for 10 minutes each, rehydrated, and then microwaved for 20 minutes with citrate buffer pH 6.0 (Sigma-Aldrich) to retrieve the epitope, as described previously (2 (link)). The sections were incubated with animal-free blocking solution (Cell Signaling Technology) for 1 hour, washed in PBS, and then incubated with primary antibodies overnight at 4°C. After 3 washes with PBS, the sections were incubated with the Signal Stain Boost Detection Reagent (Cell Signaling Technology) for 1 hour at room temperature. The tissue sections were then washed 3 times with PBS, detected using a Pierce DAB Substrate Kit (Thermo Fisher Scientific), stained with hematoxylin, and imaged by confocal microscopy. For immunofluorescent staining, the tissue sections were incubated with primary antibodies overnight at 4°C, washed, and incubated with Alexa Fluor–labeled secondary antibodies for 1 hour at room temperature. After PBS washing, both cells and tissue sections were incubated with diluted DAPI solution for 5 minutes at room temperature, mounted with an antifade mounting media, and imaged by confocal microscopy. All antibodies are listed in Supplemental Table 4 .
Animal free blocking solution
Manufactured by Cell Signaling Technology
Animal-free blocking solution is a laboratory product that can be used to block non-specific binding in immunoassays and Western blotting procedures. It is formulated without the use of any animal-derived components.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hematoxylin, by Cell Signaling Technology (2 mentions)
Signalstain dab, by Cell Signaling Technology (2 mentions)
Rabbit hrp, by Cell Signaling Technology (2 mentions)
Anti tgf β1 receptor, by Abcam (2 mentions)
Signalstain boost detection reagent hrp mouse, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce dab substrate kit, by Thermo Fisher Scientific (1 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (1 mentions)
Citrate buffer ph 6, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (1 mentions)
Zo1 1a12, by Abcam (1 mentions)
Live dead double staining kit, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Dmi4000 inverted microscope, by Leica camera (1 mentions)
9 protocols using animal free blocking solution
1
Immunohistochemistry and Immunofluorescence Staining Protocol
2
Immunofluorescent EGFR Staining in MDA-MB-231 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 cells, purchased from ATCC
(HTB-26), were cultured in Dulbecco’s Modified Eagle’s
medium (DMEM; Gibco) supplemented with 10% FBS (ATCC) and 1% penicillin/streptomycin
(Gibco) and maintained at 37 °C and 5% CO2. MDA-MB-231
cells were seeded on a 96-well plate and allowed to grow to ∼90%
confluency. Cells were then fixed with cytofix/cytoperm (BD Biosciences),
incubating for 20 min at room temperature. The fixative was removed,
and the cells were washed with MQ water (×2). Next, the cells
were blocked to prevent any nonspecific binding by incubating with
a 1× animal-free blocking solution (Cell Signaling Technology)
for 1 h at 4 °C. The blocking solution was removed, followed
by washing with MQ water (×2). The cells were then incubated
with human EGF R/ErbB1 antibody (monoclonal mouse anti-EGFR, 100 μg/mL;
R&D Systems) diluted in 8.6 mM pH 7.4 ± 0.1 monophosphate
buffer for 24 h at 4 °C. After 24 h, the cells were washed with
monophosphate buffer (×2) and next incubated with secondary antibody
(mouse IgG2B PE-conjugated, 2 μg/mL; R&D Systems)
for 2 h at 4 °C. The cells were washed with monophosphate buffer
(×2) and then visualized under fluorescent microscopy using a
Zeiss LSM 710 confocal microscope.
(HTB-26), were cultured in Dulbecco’s Modified Eagle’s
medium (DMEM; Gibco) supplemented with 10% FBS (ATCC) and 1% penicillin/streptomycin
(Gibco) and maintained at 37 °C and 5% CO2. MDA-MB-231
cells were seeded on a 96-well plate and allowed to grow to ∼90%
confluency. Cells were then fixed with cytofix/cytoperm (BD Biosciences),
incubating for 20 min at room temperature. The fixative was removed,
and the cells were washed with MQ water (×2). Next, the cells
were blocked to prevent any nonspecific binding by incubating with
a 1× animal-free blocking solution (Cell Signaling Technology)
for 1 h at 4 °C. The blocking solution was removed, followed
by washing with MQ water (×2). The cells were then incubated
with human EGF R/ErbB1 antibody (monoclonal mouse anti-EGFR, 100 μg/mL;
R&D Systems) diluted in 8.6 mM pH 7.4 ± 0.1 monophosphate
buffer for 24 h at 4 °C. After 24 h, the cells were washed with
monophosphate buffer (×2) and next incubated with secondary antibody
(mouse IgG2B PE-conjugated, 2 μg/mL; R&D Systems)
for 2 h at 4 °C. The cells were washed with monophosphate buffer
(×2) and then visualized under fluorescent microscopy using a
Zeiss LSM 710 confocal microscope.
3
Immunohistochemical Analysis of TGF-β1 and Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine protein localization and expression, the formalin-fixed tissues were de-paraffinized. After deparaffinization and dehydration of paraffin sections, heat-induced epitope retrieval was performed using a pressure cooker and sodium citrate buffer (10 mM sodium citrate, 0.05% tween-20, pH 6.0). Once boiled, slides were transferred from PBS to the sodium citrate buffer in pressure cooker for 10 min. Slides were then cooled to room temperature for 30 min, and permeabilized with permeabilization buffer containing 0.3% triton-100 in PBS for 10 min. To block endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide for 10 min and blocked with animal-free blocking solution (Cat# 15019; Cell Signaling, Inc.). After blocking, slides were incubated overnight at 4°C with mouse monoclonal anti-TGF-β1 (1:50; Cat# sc-130348; Santa Cruz, Inc.) or rabbit polyclonal anti-TGF-β1 receptor (1:100; Cat# ab235178; Abcam, Inc.) and subsequently incubated with SignalStain Boost Detection Reagent (HRP mouse; Cat# 8125; or HRP Rabbit; Cat# 8114; Cell Signaling, Inc.) for 30 min at room temperature. Slides were then incubated for 2–10 min with SignalStain DAB (Cat# 8059; Cell Signaling, Inc.), immersed in distilled H2O, stained with hematoxylin (Cat# 14166; Cell Signaling, Inc.) and mounted with coverslips. All washing steps were done three times with PBS-T (tween-20, 0.05%).
4
Immunohistochemical Analysis of p-ERK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paraffin-embedded sections were deparaffinized and dehydrated by sequential immersion in xylene and graded alcohol solutions, respectively. Sections were blocked using 1X Animal-Free Blocking Solution (Cell Signaling Technology, Inc., cat. no. 15019) at room temperature for 1 h. The sections were incubated with an antibody against p-ERK (1:100) at 4°C overnight, and subsequently incubated with a HRP-conjugated goat anti-rabbit antibody for 1 h at room temperature. The tumor sections were visualized using a DAB solution, treated with mounting reagent and observed under a routines light microscope (magnification, ×200). Finally, p-ERK positive cells were counted in three random fields from each sample.
5
TGF-β1 and Receptor Immunohistochemistry
6
Fluorescence Imaging of Tight Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For fluorescence imaging, cell samples were fixed with 4% paraformaldehyde in H2O and stored in PDMS with Ca2+ and Mg2+ ions at 4 °C. For immunofluorescence staining, the permeabilized cells (0.1% TritonX-100 in PBS; Sigma Aldrich, T8787, Vienna, Austria and Gibco, 10010023, Vienna, Austria) were labeled with a primary anti-ZO-1 monoclonal antibody (1:100 dilution; Abcam, ZO1-1A12, Vienna, Austria) and rabbit MUC1 monoclonal antibody (1:50; Abcam, EPR1023, Vienna, Austria); then the samples were blocked using animal-free blocking reagent solution (Animal-free Blocking Solution, Cell Signaling Technology, Liden, The Netherlands). For labeling, secondary Alexa Fluor 488—labelled recombinant goat anti-rabbit IgG (heavy Chain) and Alexa Fluor 555 goat anti-mouse IgG were incubated for 60 min prior to nucleic and phalloidin staining. All dyes and antibodies were diluted in PBS (Sigma-Aldrich) containing 0.5% animal-free blocking reagent (1:10 dilution) and were washed three times in between staining steps.
To determine viability, the Live/Dead Double Staining Kit from Sigma Aldrich (QIA76, Vienna, Austria) was used. An Olympus IX83 automated epifluorescence microscope was used for all microscopic imaging using a quad-bandpass filter.
To determine viability, the Live/Dead Double Staining Kit from Sigma Aldrich (QIA76, Vienna, Austria) was used. An Olympus IX83 automated epifluorescence microscope was used for all microscopic imaging using a quad-bandpass filter.
7
Immunocytochemical Characterization of iPSC-Derived Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iPSC-neurons were plated on to German-glass coverslips (Electron Microscopy services) for the terminal differentiation stage and then fixed at Day 46 of differentiation with 4% paraformaldehyde for 30 min at room temperature. Cells were permeabilized with PBS + 0.3% Triton X-100 (Sigma) for 10 min. Cells were then blocked with Animal-Free Blocking solution (Cell Signaling Technology) for 1 h at room temperature, followed by primary antibody diluted in PBS + 3% BSA (Sigma A1470) overnight at 4 °C. Primary antibodies included: MAP2 (Sigma M1406, 1:500), Tuj1 (Biolegend 801201, 1:500), PSD-95 (Cell Signaling 3450, 1:200). Cells were subsequently treated with secondary antibodies goat anti-IgG1-Alexa488 (for MAP2), goat anti-IgG2A-Alexa647 (for Tuj1), goat anti-rabbit Alexa568 (for PSD-95) (each 1:500, Invitrogen) diluted in PBS + 3% BSA for 2 h at room temp, follow by post-fixation with 4% paraformaldehyde for 15 min at room temp. Cells were counterstained with Hoechst-33342 (Invitrogen, 1:2000) in PBS for 15 min at room temperature, and mounted with Fluoromount-G (Southern Biotech). Washes with PBS were performed between each step, and a final wash with water was performed prior to mounting. Images were taken on a Leica DMi4000 inverted microscope, outfitted with a PL APO ×40 objective and Leica DFC340 FX camera.
8
Western Blot Analysis of PC4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary fibroblasts from PC4+/+ and WT mice were collected, washed and lysed with a lysis buffer (Cell Signaling Technology, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific Inc.) for 0.5 hours on ice. Total protein was quantitated with a bicinchoninic acid assay kit (Thermo Fisher Scientific Inc.). Each sample was denatured with a loading buffer (Beyotime, China), separated by electrophoresis on a 12% gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with an animal-free blocking solution (Cell Signaling Technology) and incubated with PC4 primary antibody (11 000, ab84459, Abcam) for 16 hours at 4°C. The membranes were washed thrice with Tris-Buffered Saline Tween-20 (TBST) (5 minutes each wash) and incubated for 1–2 hours with horseradish peroxidase-linked secondary antibody (Beyotime) at 25°C. The intensity of bands was visualized and determined using an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Images were quantified with ImageJ software. Actin was used as a loading control. PC4/actin shows relative expression level of PC4 protein.
9
Immunohistochemical Staining of Mouse Osteomodulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Epitope retrieval was performed using chondroitinase ABC (50 units per mL, Sigma-Aldrich) in 60 mmol·L−1 sodium acetate and 100 mmol·L−1 Tris (pH 8) for 30 min at 37 °C. Animal-free blocking solution (Cell Signaling Technology, dilution 5X) was used to block the sections prior to overnight incubation with the primary polyclonal goat antibody anti-mouse OMD (R&D Systems, AF3308, 0.8 μg·mL−1) in antibody diluent (Dako, S2022). The sections were then incubated for 30 min with the secondary polyclonal rabbit antibody anti-goat coupled with HRP (DakoP0449, dilution 1:400) diluted in Antibody diluent. Visualization of the secondary antibody was performed by using DAB (Cell Signaling Technology, 8059) for 2 min. Sections were counterstained with hematoxylin from Carazzi (Sigma-Aldrich) for 4 min.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!